![Figure 2. Effect of anti–TIM-1, anti–TIM-3, or rmTIM-4 on mast-cell function. C57BL/6J BMCMCs were sensitized with anti-DNP IgE (H1-ϵ-26) at 37°C overnight and then washed. Naive or IgE-sensitized C57BL/6J BMCMCs were cultured in the presence or absence of Ag (DNP-HSA, 20 ng/mL) together with (A) 20 μg/mL anti-TIM Abs or control IgG or 20 μg/mL rmTIM-4 at 37°C for 1 hour for β-hexosaminidase (β-Hex) release assay (P +I = PMA [0.1 μg/mL] + Ionomycin [1.0 μg/mL]); (B) 20 μg/mL anti-TIM Abs or control IgG or 20 μg/mL rmTIM-4 at 37°C for 6 hours for cytokine production; (C) 0 to 100 μg/mL rmTIM-4 at 37°C for 6 hours for cytokine production in IgE/Ag-stimulated cells; (D) 40 μg/mL rmTIM-4 and 80 μg/mL anti–TIM-4 pAb or control IgG (goat IgG) at 37°C for 6 hours for cytokine production in IgE-sensitized cells ± Ag; (E) 40 μg/mL rmTIM-4 and 0 to 40 μg/mL rmTIM-1 at 37°C for 6 hours for cytokine production in IgE/Ag-stimulated cells; (F) 20 μg/mL anti-TIM Abs or control IgG or 20 μg/mL rmTIM-4 at 37°C for 0, 1, 2, 4, or 6 days in the absence of IL-3 for apoptosis (the same data for culture with IL-3, rat IgG and goat IgG, or without any reagents [medium] are shown in the top and bottom panels for each group of BMCMCs); (G) 20 μg/mL anti-TIM Abs or control IgG at 37°C for 2 days for IL-3 production; (H,I) 20 μg/mL anti-TIM Abs or control IgG at 37°C for 2 days in the absence of IL-3 for Bcl-2 expression (the FACS data shown are representative of those obtained in 3 experiments using 3 different batches of BMCMCs). Top numbers indicate MFI for anti–Bcl-2 mAb staining; bottom numbers, MFI for control IgG staining. “IL-3” indicates results for BMCMCs maintained in IL-3–containing WEHI-3 cell-conditioned medium. (A-G, I) *P < .05 versus corresponding values for cells cultured with control IgG (B,D,F,G) or with no rmTIM-4 or rmTIM-1 (0 μg/mL) (C,E). Data shown in panels A to G and I are the average ± SEM (n = 3) of duplicate values for each condition obtained from an experiment done using 3 different batches of BMCMCs; such experiments were done 2 to 4 times each, and each of these gave similar results.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/110/7/10.1182_blood-2006-11-058800/2/zh80210708280002.jpeg?Expires=1735569006&Signature=hLJYWbBjjCxe3DKSKsfrORLbs~zvwpHzOr~-SJ384wiV9NtzsNGjB9kSOPh6rnbqtRIkmD8X1AfriSkCAcjnqkmfHbOcDUXaK~xgUthDVuARbPFILJXPWA2lLzkLy5Y6xSOR-tg30egzZdeihJUZqbvLUtUhwGwIES~4wBeYI4CfINbb6ebsc8-ueNyMJq~vSGj691pQow69vVQkRSn4XzLj5~Are4UY9za6tvlvrSpLcDo~FW5QFpNL7kkgeXT0d2wqcaQVPoFKedsLnFJhVrDXOeOCfCbS8~VfLvTg4H3mEOzZ29886wDisgdvTHry7Zfl72gTvMeR4Z8LD9377A__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Effect of anti–TIM-1, anti–TIM-3, or rmTIM-4 on mast-cell function. C57BL/6J BMCMCs were sensitized with anti-DNP IgE (H1-ϵ-26) at 37°C overnight and then washed. Naive or IgE-sensitized C57BL/6J BMCMCs were cultured in the presence or absence of Ag (DNP-HSA, 20 ng/mL) together with (A) 20 μg/mL anti-TIM Abs or control IgG or 20 μg/mL rmTIM-4 at 37°C for 1 hour for β-hexosaminidase (β-Hex) release assay (P +I = PMA [0.1 μg/mL] + Ionomycin [1.0 μg/mL]); (B) 20 μg/mL anti-TIM Abs or control IgG or 20 μg/mL rmTIM-4 at 37°C for 6 hours for cytokine production; (C) 0 to 100 μg/mL rmTIM-4 at 37°C for 6 hours for cytokine production in IgE/Ag-stimulated cells; (D) 40 μg/mL rmTIM-4 and 80 μg/mL anti–TIM-4 pAb or control IgG (goat IgG) at 37°C for 6 hours for cytokine production in IgE-sensitized cells ± Ag; (E) 40 μg/mL rmTIM-4 and 0 to 40 μg/mL rmTIM-1 at 37°C for 6 hours for cytokine production in IgE/Ag-stimulated cells; (F) 20 μg/mL anti-TIM Abs or control IgG or 20 μg/mL rmTIM-4 at 37°C for 0, 1, 2, 4, or 6 days in the absence of IL-3 for apoptosis (the same data for culture with IL-3, rat IgG and goat IgG, or without any reagents [medium] are shown in the top and bottom panels for each group of BMCMCs); (G) 20 μg/mL anti-TIM Abs or control IgG at 37°C for 2 days for IL-3 production; (H,I) 20 μg/mL anti-TIM Abs or control IgG at 37°C for 2 days in the absence of IL-3 for Bcl-2 expression (the FACS data shown are representative of those obtained in 3 experiments using 3 different batches of BMCMCs). Top numbers indicate MFI for anti–Bcl-2 mAb staining; bottom numbers, MFI for control IgG staining. “IL-3” indicates results for BMCMCs maintained in IL-3–containing WEHI-3 cell-conditioned medium. (A-G, I) *P < .05 versus corresponding values for cells cultured with control IgG (B,D,F,G) or with no rmTIM-4 or rmTIM-1 (0 μg/mL) (C,E). Data shown in panels A to G and I are the average ± SEM (n = 3) of duplicate values for each condition obtained from an experiment done using 3 different batches of BMCMCs; such experiments were done 2 to 4 times each, and each of these gave similar results.
